Chlamydia species are important causes of human disease for which no vaccine exists. An important gap in our knowledge is how this obligate intracellular parasite establishes a privileged niche--a membrane bound compartment termed the inclusion--in order to survive and replicate in the hostile intracellular environment. Chlamydiae encode a distinctive family of secreted effectors, the Incs (Inclusion membrane proteins) that are translocated from the bacteria and inserted into the inclusion membrane. We hypothesize that these effectors are ideally positioned at the host-pathogen interface to mediate interactions between the inclusion and the host and contribute to successful intracellular survival. This grant builds on our extensive preliminary studies in which we used large-scale affinity purification/mass spectrometry (AP-MS) to comprehensively identify protein- protein interactions (PPIs) between all C. trachomatis Incs and the human proteome. From amongst 404 interactions for 38/62 Incs, we identified a plethora of new potential interactions. We propose to use biochemical, cell biological, and newly developed Chlamydial genetic strategies to validate our highest priority Inc-host PPIs and explore their role in C. trachomatis infections. In Aim 1, we follow-up at the detailed molecular level our novel observation that IncE subverts retromer components and possibly a subset of syntaxins to modulate host cell vesicular trafficking. In aim 2, we will investigate the mechanism and functional significance of the interaction of the Inc CT192 with the dynactin complex. We propose that either CT192 sequesters dynactin to interfere with dynein-dependent transport or that it allows the inclusion to hitch a ride onto dynein-dependent microbule transport. The proposed approaches are applicable to other high confidence PPIs that we have identified and determined to be high priority. In aim 3, we team up with our Co-investigator, Dr. Nevan Krogan, to apply powerful new proteomic technologies to globally profile changes in the host ubiquitome in response to pathogens. Our finding that up to 12 Incs appear to interact with various components of the host cell ubiquitin machinery suggests that Chlamydia reprograms the host ubiquitin program to facilitate infection. We prioritized study of Inc CT383, as it is expressed early, predicted to interact with 3 different Ub ligases, and has the potential to substantially remodel the host ubiquitinome. Together, these aims build upon our extensive preliminary data and allow us to comprehensively understand how Chlamydia employs Incs to create a unique intracellular niche and reprogram the host.